The present invention involves using a new technique for the detection of genomic abnormalities. Such detection may prove particularly useful to predict recurrence of cancer, or early detection of cancer.
High frequency of recurrence is one of the major problems in cancer treatment. Relapse from clinically undetectable residual disease is the most likely mechanism (1). Detection of minimal disease is extremely difficult since tumor specific markers are not readily available. Molecular technology has provided a means to demonstrate residual disease by identifying clonal gene rearrangement patterns that may present, for example, in malignant hematopoietic cells (2). Southern blot hybridization may detect neoplastic cells at levels as low as 1% of the total number of cells (3). However, one of the major drawbacks in using such traditional methods is that it is difficult to be certain that faint nongermline bands indeed represent clonal rearrangements representative of neoplastic cells. Furthermore, no rearranged bands can usually be detected by traditional methods in cases in which the concentration of neoplastic cells is below 1%. It is expected that such a low concentration occurs frequently while patients are in remission.
Saiki et al. have recently utilized a new technique, sequence amplification by polymerase chain reaction (PCR), to diagnose sickle cell anemia prenatally (4). This technique is highly sensitive. It requires small amounts of DNA (less than 1 ug) and can amplify copies of target DNA sequences exponentially.
Recently, the present inventor has utilized polymerase chain reaction (PCR) technology to preferentially amplify the hybrid DNA sequences of a chromosomal translocation, t(14;18), characteristic of follicular lymphoma. This technique detected neoplastic cells carrying the t(14;18) in a concentration of less than 1:100,000 (5).
A major limitation of the PCR technique has been thought to involve the ability to amplify only short DNA segments (e.g., several hundred base pairs). Such amplification was feasible for the t(14;18) translocation because the molecular breakpoints on both chromosomes occurred within a limited region (6-8). However, this approach was not applicable to chromosomal translocations with more variable breakpoints, and also was not feasible for amplification of RNA without major modifications of the procedure. The present invention relates to modification of the PCR technique so that it may detect the presence of minimal amounts of aberrant gene transcripts from the neoplastic cells that carry a chromosomal translocation with variable molecular breakpoints. Neoplastic cells such as those bearing the Philadelphia Ph.sup.1 chromosome, for example, could then be detected.
Ph.sup.1 chromosome (t(9;22) (q34;q11)), has been observed in more than 90% of chronic myelogenous leukemia (CML) cases (9,10). Even though the breakpoints on chromosome 22q11 cluster within a small DNA segment designated as bcr (11,12), the breakpoints on chromosome 9q34 occur at variable positions up to more than 100 kilobases (kb) 5' to the second exon of the c-abl oncogene (13,14). Despite the variability of the molecular breakpoints at the DNA level, the fused bcr/abl gene in CML is transcribed into two types of chimeric mRNA: one with abl exon II linked to bcr exon "2" (designated as L-6 junction) and the other one with abl exon II linked to bcr exon "3" (designated as K-28 junction) (15). By means of RNase protection analysis, the Ph.sup.1 -positive K562 cell line has been shown to have both types of mRNA (15). In a study of 21 Ph.sup.1 -positive CML patients, one or both RNA junctions were detected in 19 patients (16).